Our research interests are to map and isolate novel cancer-causing genes, to clarify their normal functions and their relationship to environmental carcinogenesis. To facilitate this process, we have developed human gene targeting system using recombination-proficient chicken DT40/human cell hybrids. Any human gene or chromosome in chicken DT40 cells can be targeted and modified at high frequency. We have developed the following three applications of this system; 1) a novel yeast artificial chromosome (YAC) cloning method which allows one to isolate a specific region of DNA segment by gene targeting; 2) a system which allows one to assess the functions of a specific gene by disruption of the gene (gene knock-out); 3) a system which allows one to create a truncated transferrable chromosome at specific gene locus by targeted insertion of a telomere sequence. One of our research projects has been directed toward identifying a novel cellular senescence gene on human chromosome 1 whose introduction into the human osteosarcoma cell line, TE85, induces growth arrest. To localize a gene to a clonable size of the chromosomal region (less than 1 Mb), we plan to perform genetic complementation studies by transferring decreasing sized genetic units, including whole chromosomes, truncated chromosomes, subchromosomal fragments, YAC clones and BAC clones, to TE 85 cells. To facilitate this process, we have generated mouse A9 hybrid cell lines containing a transferable human chromosome 1 truncated at various sites of this chromosome. We are currently transferring these chromosomes in A9 cells to TE85 to map the growth arrest activity to a region of 40 Mb on chromosome 1q. To further narrow down the senescence activity, we plan to develop a minichromosome or an artificial chromosome carrying a genetic unit whose size is larger than a cosmid clone but smaller than a chromosomal fragment. This will facilitate gene mapping as well as functional analysis of a single gene. The minichromosome derived from a human chromosome 11 will be generated in DT40 by targeted insertion of a telomere sequence into the centromere of this chromosome. Also, the centromere region of chromosome 11 has been isolated into YAC clones and will be used for constructing a YAC based artificial chromosome. To determine the functions of the human mismatch repair gene, hMSH2, we have generated transferrable human chromosome 2 with a modified hMSH2 in DT40 cells. Modified chromosome 2 will be transferred into the hMSH2-defective cell line, LoVo, to determine the functions of the hMSH2. - gene targeting, homologous recombination, gene knock-out, yeast arteficial chromosomes, telomere